Dual catalytic hairpin assembly-powered DNA walker activation of CRISPR/Cas12a for label-free electrochemical detection of hepatocellular carcinoma biomarkers
Guoyu Liu, Li Yang, Huiming Li, Xiaoli Xiong, Yao Gao, Tao Zhang, T.‐L. LIU, Xiaochun Wu, Liping Zhu
Abstract
Early and accurate diagnosis of hepatocellular carcinoma (HCC) requires ultrasensitive and universal strategies targeting low-abundance biomarkers. Here, an electrochemical biosensor was developed for detecting HCC biomarkers miRNA-122 and AFP, featuring a DNA tripod-supported interface integrated with dual catalytic hairpin assembly (DCHA)-DNA walker cascade amplification and CRISPR/Cas12a signal transduction. The target triggered the CHA1 reaction to generate H1/H2 complexes, which functioned as DNA walkers to initiate the CHA2 reaction on magnetic bead surfaces and released H3/H4 products containing protospacer adjacent motif (PAM) sequences. CRISPR/Cas12a recognized the PAM sequence and nonspecifically cleaved ssDNA on the DNA tripod, which inhibited the formation of G-quadruplex/hemin complexes and reduced the electrochemical signal, thereby achieving label free detection of target. The spatially confined DCHA-DNA walker cascade reaction on magnetic bead surfaces significantly enhanced reaction efficiency, while the DNA tripod structure improved probes orientation, hemin binding efficiency, and CRISPR/Cas12a accessibility to signal probes, ensuring ultra-high sensitivity and stability of the biosensor. This platform exhibits high programmability, requiring only simply encoding of the target recognition process to sensitively detect AFP and miRNA-122, with detection limits of 18.89 fg/mL and 34.02 aM (S/N = 3), respectively. Notably, the biosensor shows excellent applicability in real serum samples and effectively distinguished between healthy individuals and HCC patients, demonstrating its promising application prospects in the early and accurate diagnosis of HCC.